JP4840906B2 - Method for producing liposome using lipid pattern - Google Patents

Description

  The present invention relates to a method for producing a liposome using a lipid pattern, and more particularly to a method for producing a monodisperse giant liposome using a repeat lipid pattern.

  As shown in FIG. 6, the liposome is a spherical vesicle including an aqueous solution composed of a bilayer mainly composed of phospholipid, and has a structure similar to that of the cell membrane. In FIG. 6, the liposome 100 includes a lipid bilayer 101, and the bilayer 101 includes a hydrophilic head 102 and a hydrophobic tail 103.

  Among the liposomes having such a structure, in recent years, the application of “giant” liposomes having a diameter as large as 10 to 100 microns has attracted attention (see Non-Patent Document 1). Giant liposomes have the greatest advantage that they can be observed directly under a microscope due to their size (see Non-Patent Document 2), and are the same size as cells and can be used as models of artificial cells (non-patents) (See Patent Document 3). Furthermore, drugs and DNA can be encapsulated in liposomes, and can also be used as micro-sized containers (see Non-Patent Document 4).

  FIG. 7 is a diagram showing a liposome in which a protein is incorporated into a membrane, and FIG.

As shown in FIG. 7, it is considered that a device that individually analyzes the behavior of various membrane proteins can be realized by introducing the membrane protein 104 into the liposome membrane. For example, the drug transport efficiency can be elucidated by applying a drug to the liposome containing the membrane protein from the outside and measuring the amount taken into the liposome as the concentration change in the liposome by the fluorescence intensity.
Liisi, P.A. L. , Giant Vehicles, pp. 3-9 (2000) Angelova, M .; I. , Giant Vehicles, pp. 27-36 (2000) Y. Yoshikawa, Chemical Physics Letters, 366, pp. 305-310 (2002) G. Trese, Analytical Chemistry, (in press) B. Ilic and H.M. G. Craighead, Biomedical microdevices, pp 317-322, 2000 R. N. Ortho et al Biophysical Journal Vol. 85, November 2003. 3066-3073

  However, liposomes prepared by the conventional method have various sizes as shown in FIG. 8 (a), and if the internal volume is different, the concentration change per unit time is also different (the larger the concentration, the less the concentration change). == low fluorescence brightness) As a result, the uptake of chemicals has to deal with ambiguous data with large dispersion.

  Under such circumstances, in this field, there is a strong demand for a method for efficiently producing liposomes having uniform volumes (uniform diameters) and uniform shapes (FIG. 8B).

  In addition, liposomes prepared by the conventional method have various sizes as shown in FIG. 8 (a). If no post-treatment is performed such as passing through a filter to make the particle sizes uniform, a liposome having a uniform diameter can be obtained. I can't. In addition, a technique for patterning a phospholipid film by using a stamp made of PDMS (polydimethylsiloxane) made of silicone rubber by Taylor et al. (P. Taylor and et al., PCCP, pp4918-4922, 2003) and electro Attempts have been made to produce uniform liposomes using forming, but the PDMS stamp expands when an organic solvent is applied, and the stamp is distorted when pressed, making it impossible to produce liposomes of the desired size. Disadvantages have been pointed out (see Non-Patent Document 5 above).

  Also, a parylene resin sheet is used in place of the PDMS stamp (Non-Patent Document 6 below), but there is no indication of using this to produce liposomes from lipid solutions by electroformation. It has not been done.

  In view of the above circumstances, the present invention provides a method for producing liposomes using a lipid pattern, which can efficiently form uniform liposomes from a lipid film having a uniform shape by electroformation. For the purpose.

In order to achieve the above object, the present invention provides
[1] In a liposome production method using a lipid pattern, an expansion-resistant polymer resin sheet in which a plurality of uniform holes are formed on a conductive substrate is prepared, and a lipid solution is placed on the expansion-resistant polymer resin sheet. Applying and peeling the expansion resistant polymer resin sheet from the substrate to form a patterned lipid film of uniform shape on the substrate, and having the patterned lipid film of uniform shape Fill the chamber in which the conductive substrate is the bottom electrode and the upper electrode made of the conductive substrate is disposed on the top via the insulating spacer with an aqueous solution, and an electric field is applied between the upper electrode and the bottom electrode to form a uniform shape. It is characterized by forming a liposome.

  [2] The liposome production method using the lipid pattern according to [1] above, wherein the expansion-resistant polymer resin sheet is a parylene resin sheet.

  [3] In the method for producing liposomes using the lipid pattern according to [1], the conductive substrate is an ITO substrate.

  [4] In the method for producing a liposome using the lipid pattern according to [1], the insulating spacer is silicone.

  [5] The method for producing a liposome using the lipid pattern according to [1] above, wherein the uniform liposome is a giant liposome having a diameter of 10 to 100 μm.

[6] In the method for producing a liposome using the lipid pattern according to [1] above, in order to apply the electric field, 0.5 to 2.5 V _pp at 10 Hz between the upper electrode and the bottom electrode. An alternating current is applied.

  [7] The liposome production method using the lipid pattern according to [1] above, wherein a deviation coefficient of the liposome size is 1 to 15%.

  [8] The method for producing liposomes using the lipid pattern according to [1] above, wherein a plurality of uniform holes are formed in the expansion-resistant polymer resin sheet by lithography.

  According to the present invention, the following effects can be achieved.

  (1) Uniform liposomes can be efficiently formed from a patterned lipid film having a uniform shape by electroformation.

  (2) Giant liposomes that are uniform and have a diameter of 10-100 μm can be produced.

  In the method for producing liposomes using the lipid pattern of the present invention, an expansion-resistant polymer resin sheet in which a plurality of uniform holes are formed on a conductive substrate is prepared, and a lipid solution is placed on the expansion-resistant polymer resin sheet. Apply, peel off the expansion resistant polymer resin sheet from the substrate to form a uniform shaped patterned lipid film, and use the conductive substrate with the uniform shaped patterned lipid film as the bottom electrode Then, the inside of the chamber in which the upper electrode made of a conductive substrate is disposed on the upper portion through an insulating spacer is filled with an aqueous solution, and an electric field is applied between the upper electrode and the bottom electrode to form a uniform liposome.

  Hereinafter, embodiments of the present invention will be described in detail.

  FIG. 1 is a schematic diagram of a liposome production apparatus using a lipid pattern showing an embodiment of the present invention, and FIG. 2 is a diagram showing a production process of a uniform size liposome using the same, in which a parylene resin sheet pattern is shown. A forming method will be described.

  FIG. 1 shows a device for making uniform diameter liposomes. In this figure, two ITO glass plates 1 and 8 are used as electrodes, and a chamber 9 is formed separated by a silicone spacer 7. The inside of the chamber 9 is filled with degassed water (but it may be a normal aqueous solution). Further, an AC voltage is applied from the power source 10 to the chamber 9 through the two ITO glass plates 1 and 8 to form uniform-sized giant liposomes (10 to 100 μm).

  (1) First, as shown in FIG. 2A, a parylene resin as an expansion-resistant polymer resin sheet is vapor-deposited on the ITO glass plate 1, and the parylene resin sheet 2 is laminated.

(2) Next, as shown in FIG. 2 (b), the parylene resin sheet 2 on the ITO glass plate 1 is patterned with O ₂ plasma to produce a perforated sheet 3. At that time, the hole 4 of the perforated sheet 3 is set to have a predetermined area accurately.

  (3) Next, as shown in FIG. 2 (c), a lipid solution 5 made of a solution obtained by mixing phospholipid, chloroform and methanol is applied onto the perforated sheet 3 in which the holes 4 having a predetermined area are formed. And dried in vacuum.

  (4) Next, as shown in FIG. 2 (d), when the perforated sheet 3 with the holes 4 having a predetermined area is peeled off, the lipid pattern 6 having a predetermined area is transferred to the ITO glass plate 1. Remain. The ITO glass plate 1 is disposed at the lower portion, and the ITO glass plate 8 is disposed at the upper portion through the silicone spacer 7.

  (5) Next, as shown in FIG. 2 (e), when degassed water 11 is introduced into the chamber 9 for liposome production and an electric field is applied to the ITO glass plates 8, 9 by electroformation, Within 9, single-sized giant liposomes 12 are produced.

  As described above, uniform-shaped liposomes can be efficiently formed from a patterned lipid film having a uniform shape by electroformation.

  FIG. 3A shows a fluorescent image of 20 μm □ liposomes. FIG.3 (b) is a figure which shows intensity distribution along the AB line | wire of a lipid pattern, The clear edge of the patterned lipid film on an ITO glass plate can be confirmed from the intensity profile of this pattern.

  FIG. 4 shows the steps in the experiment.

  FIG. 4 (a) shows a patterned lipid film of 50 × 50 μm square, and FIG. 4 (b) shows a patterned lipid film expanded by applying an electric field, FIG. 4 (c). Shows a state in which giant liposomes are formed. As a comparative example, FIG. 4 (d) shows small liposomes formed from a 20 × 20 μm square patterned lipid film.

  FIG. 5 is a view showing the size distribution of liposomes formed by the present invention and the conventional method. FIG. 5 (a) shows the size distribution of liposomes when liposomes are formed without using a patterned lipid film. Here, liposome sizes are distributed over a wide range, and the deviation coefficient is 70%. And high. In contrast, when the patterned lipid film of the present invention is used, the deviation coefficient is 15%, which is a significantly low deviation coefficient. As a result of further development, the deviation coefficient could be improved to the 1% level. Thereby, it turns out that the size of the liposome formed by this invention is substantially uniform.

  In addition, this invention is not limited to the said Example, Based on the meaning of this invention, a various deformation | transformation is possible and these are not excluded from the scope of the present invention.

  The method for producing liposomes using the lipid pattern of the present invention is suitable for producing giant liposomes having the same structure as the cell membrane.

It is a schematic diagram of the manufacturing apparatus of the liposome using the lipid pattern which shows the Example of this invention. It is a figure which shows the manufacturing process of the liposome of uniform size using the manufacturing apparatus of the liposome using the lipid pattern which shows the Example of this invention. It is a figure which shows the fluorescence image of the 20 micrometers square liposome concerning this invention. It is a figure which shows the process in experiment of this invention. It is a figure which shows distribution of the size of the liposome formed by this invention and the conventional method. It is a schematic diagram of a liposome having a bilayer membrane. It is a figure which shows the integration state of the membrane protein to the liposome with a double membrane. It is a schematic diagram of the liposome which takes in a chemical | medical agent.

Explanation of symbols

1,8 ITO glass plate 2 Parylene resin sheet 3 Perforated sheet 4 Hole 5 Lipid solution 6 Lipid pattern of predetermined area 7 Silicone spacer 9 Chamber 10 Power supply 11 Deaerated water 12 Single size giant liposome

Claims (8)

(A) preparing an expansion-resistant polymer resin sheet in which a plurality of uniform holes are formed on a conductive substrate;
(B) applying a lipid solution on the expansion resistant polymer resin sheet;
(C) forming a patterned lipid film having a uniform shape on the substrate by peeling the expansion-resistant polymer resin sheet from the substrate;
(D) The conductive substrate having the uniform shaped patterned lipid film is used as the bottom electrode, and the inside of the chamber in which the upper electrode made of the conductive substrate is disposed on the upper portion through the insulating spacer is filled with the aqueous solution.
(E) applying an electric field between the top electrode and the bottom electrode;
(F) A method for producing a liposome using a lipid pattern, characterized by forming a liposome having a uniform shape.   The method for producing a liposome using a lipid pattern according to claim 1, wherein the expansion-resistant polymer resin sheet is a parylene resin sheet.   The method for producing a liposome using a lipid pattern according to claim 1, wherein the conductive substrate is an ITO substrate.   The method for producing a liposome using a lipid pattern according to claim 1, wherein the insulating spacer is silicone.   The method for producing a liposome using a lipid pattern according to claim 1, wherein the uniform shaped liposome is a giant liposome having a diameter of 10 to 100 µm. 2. The method for producing a liposome using a lipid pattern according to claim 1, wherein an alternating current of 0.5 to 2.5 V _pp is applied at 10 Hz between the upper electrode and the bottom electrode in order to apply the electric field. The manufacturing method of the liposome using the lipid pattern characterized by the above-mentioned.   The method for producing a liposome using a lipid pattern according to claim 1, wherein the liposome has a size deviation coefficient of 1 to 15%.   The method for producing a liposome using a lipid pattern according to claim 1, wherein a plurality of uniform holes are formed by lithography in the expansion-resistant polymer resin sheet.